The self-association of amphotericin B and hamycin, the heptaene polyene antibiotics, was observed in alcoholic solutions on addition of increasing amounts of water at a studied antibiotics concentration of 1 10 - 5 M. An increase in the aggregation was noticed in methanolic solutions at and above 65-70% (vol. %) water content. The antibiotics were found to be aggregated at and above 85% water content in propanol. The increased water content required to initiate the aggregation in propanol was related to the more hydrophobic nature of the latter. The aggregation of antibiotics was hindered by the addition of Triton X-100, a non-ionic detergent, at a concentration of 0.5%. The self-association of amphotericin B and hamycin was observed at concentrations of 5 10 - 7 and 1 10 - 7 M respectively, in phosphate-buffered saline as detected by the presence of an excitonic doublet in their circular dichroism (CD) spectra. The aggregation was found to be enhanced while the concentration increased up to the studied concentration of 5 10 - 5 M. The polyene antibiotics existed as monomers in dimethyl sulfoxide(DMSO) at concentrations of 1 10 - 7 -4.3 10 - 2 M and 1 10 - 7 -1 10 - 2 M for amphotericin B and hamycin respectively, as revealed by their concentration-independent CD spectra.